Project Summary/Abstract The goal of the proposed clinical study is to induce tolerance to combined kidney and hematopoietic cell transplants after conditioning with total lymphoid irradiation (TLI) and anti-thymocyte globulin (ATG) in patients given grafts from related living HLA haplotype matched donors or from unrelated living donors who are matched at 3 HLA antigens. Establishment of tolerance is expected to eliminate the usual lifelong need for immunosuppressive (IS) drugs and attendant side effects such that complete IS drug withdrawal will be accomplished without subsequent evidence of rejection. The proposed studies build upon our progress in the previous grant periods in the successful induction of chimerism and tolerance in about 80% of fully HLA matched patients, the ability to establish persistent high levels of mixed chimerism in related HLA haplotype matched patients, and the 100% kidney graft survival in 25 HLA matched and 20 HLA mismatched patients enrolled in the tolerance protocols during the past 15 years. We will test the hypothesis that high levels of mixed chimerism that persist for more than 1 year will be maintained during and after IS drug withdrawal, and result in tolerance. We hypothesize that tolerance will be predicted by evidence of alloreactive T cell clonal deletion measured by high throughput sequencing of TCR genes, lack of development of donor specific antibodies (DSA), and lack of inflammatory cell PCR products in the urine. The changes in the lymphoid tissues that promote tolerance after conditioning will be monitored by determining changes in the balance of effector T cells and immunosuppressive regulatory cells including monocytes and myeloid derived suppressor cells (MDSCs) in the blood. In order to assess the longterm outcome of patients, we propose to establish a registry of efficacy and risks for all patients enrolled in our tolerance protocols since inception for comparison to standard of care patients in our centers and in the national registry. In addition, we will perform longterm immune monitoring parameters to measure persistence of chimerism, donor specific unresponsiveness in the MLR, and the T cell clonal repertoire. In conclusion, we will attempt to induce tolerance, identify mechanisms and predictors such that IS drugs can be guided appropriately, and determine the durability of immune changes that underlie tolerance.